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J Biol Chem, Vol. 275, Issue 18, 13411-13414, May 5, 2000

Ca²⁺ Depletion and Inositol 1,4,5-Trisphosphate-evoked Activation of Ca²⁺ Entry in Single Guinea Pig Hepatocytes^*

Gilles Guihard, Jacques Noel^§, and Thierry Capiod^¶

From INSERM U442, Université Paris-Sud, Bât. 443, 91405 Orsay, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Store-operated Ca²⁺ entry was investigated by monitoring the Ca²⁺-dependent K⁺ permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca²⁺ stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca²⁺-Mg²⁺-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca²⁺ store depletion on Ca²⁺ influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP₃) release from caged InsP₃ after treatment of the cells with 2,5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca²⁺-free solutions. We show that the photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP₃, and Ca²⁺ store depletion have additive effects to activate a high level of Ca²⁺ entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP₃ receptors and Ca²⁺ channels in the plasma membrane, although the nature of these Ca²⁺ channels in hepatocytes is unclear.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Ca²⁺ signals evoked by an InsP₃-dependent¹ agonist consist of two phases, Ca²⁺ release from intracellular stores and Ca²⁺ influx across the plasma membrane (1). Ca²⁺ entry is regulated by the Ca²⁺ content of the stores, a type of behavior referred to as store-operated Ca²⁺ entry or capacitative Ca²⁺ entry (2). The nature of the coupling between these two events is unclear, but evidence is accumulating that there is a direct link between InsP₃ receptors and plasma membrane Ca²⁺ channels (3, 4). The Ca²⁺ release-activated Ca²⁺ channels have yet to be cloned, but the transient receptor potential (TRP) seems to interact with InsP₃ receptors to open a path for Ca²⁺ (5). However, the existence of a TRP in adult hepatocytes has yet to be demonstrated (6). Ca²⁺-mobilizing hormones stimulate ⁴⁵Ca uptake in hepatocytes (7, 8), which may occur via two separate pathways (9-11). Ca²⁺ entry pathways, such as Ca²⁺ release-activated Ca²⁺ currents (I_crac) (12), present in hepatocytes (13) may contribute to the sustained plateau phase observed in response to Ca²⁺-mobilizing agonists.

The store-operated Ca²⁺ currents (14) are much smaller than the currents associated with the Ca²⁺-dependent conductances in guinea pig hepatocytes (15), rendering them undetectable in normal physiological conditions. Guinea pig hepatocytes possess a Ca²⁺-dependent K⁺ conductance that has proved useful for monitoring the variations in [Ca²⁺]_i near the plasma membrane (15, 16). This conductance has been well characterized and is a good alternative to whole cell Ca²⁺ measurements with fluorescent Ca²⁺ dyes. It shows no voltage dependence and no desensitization for Ca²⁺, and it is not directly activated by InsP₃ itself (17). Thus, in voltage clamped guinea pig hepatocytes, voltage steps to positive membrane potentials can be used to test the dependence of the K⁺ current on external calcium because store-operated Ca²⁺ entry is almost abolished at +40 mV (12, 18).

Although the discharge of the intracellular Ca²⁺ stores in physiological conditions occurs after agonist stimulation, the depletion of Ca²⁺ stores can be achieved using specific blockers of the intracellular Ca²⁺-Mg²⁺-ATPases such as 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) (19) and thapsigargin (20). In this work, we investigated the effect on Ca²⁺ entry of depleting internal Ca²⁺ stores in single hepatocytes using InsP₃ release from caged InsP₃ if InsP₃-dependent agonists did not mobilize internal Ca²⁺ in Ca²⁺-free solutions. We found that InsP₃ and store depletion have additive effects to activate Ca²⁺ influx in single guinea pig hepatocytes. This suggests that there is a direct functional interaction between InsP₃ receptors and Ca²⁺ channels in the plasma membrane (5), although the nature of these Ca²⁺ channels in hepatocytes is unknown.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Preparation-- Guinea pig (males of the Hartley strain) liver cells were isolated by collagenase digestion and mechanical dispersion (21). Thirty min after preparation, hepatocytes were plated in 35-mm diameter Falcon plastic Petri dishes at a density of approximately 400,000 cells in 2 ml of Earle's minimal essential medium (Life Technologies, Inc.) supplemented with fetal calf serum (10%), penicillin (200,000 units/ml), and streptomycin (100 µg/ml). The dishes were incubated at 37 °C in an atmosphere of 5% CO₂ in air for at least 2 h and were then used within 8 h. Recordings were made at a temperature of 32-34 °C.

Patch Clamp Recording-- Standard tight seal whole-cell recording techniques were used (22). Patch pipette resistances were typically 5 megaohms. Whole-cell currents and potentials were measured with a RK300 patch clamp amplifier (Biologic, Grenoble, France). Signals from whole-cell recordings were digitized using a CED 1401 interface (CED Ltd., Cambridge, United Kingdom), and traces were treated with the VCAN package supplied by J. Dempster (University of Strathclyde, UK). Experiments were carried out in chloride-free conditions with gluconate used to replace chloride. The external solution contained 145 mM sodium gluconate, 5.6 mM potassium gluconate, 5 mM CaSO₄, 1.2 mM MgSO₄, 0.4 mM NaH₂PO₄, 8 mM Hepes (pH 7.3). Patch pipettes contained 153 mM potassium gluconate; 3 mM ATPNa₂, 3 mM MgSO₄, 8 mM Hepes (pH 7.3). For the extracellular Ca²⁺-free solution, CaSO₄ was omitted, MgSO₄ concentration was increased to 2.5 mM, and 40 µM EGTA was added to the perfusing solution. The dish was perfused continuously, and hormones or drugs were added to the external medium. All solutions were 0.22-µm Millipore-filtered.

Flash Photolysis-- Caged InsP₃ or caged 5-thio-InsP₃ was introduced into the cell via the patch pipette as described previously (17). Photolysis was achieved using a 1-ms pulse from a xenon arc flashlamp (23) focused on an area of about 2 × 3 mm around the cell from an incident angle of 38° to the horizontal. This arrangement minimized (about 8%) energy loss due to reflection from the fluid surface. Light was band pass-filtered (300-350 nm) with a UG 11 filter. Photolytic conversion of caged ATP was assessed by high performance liquid chromatography, and the value was used to calculate the photolytic conversion of caged InsP₃ and caged 5-thio-InsP₃ as described previously (17, 24).

Chemicals-- Pure caged InsP₃ and caged 5-thio-InsP₃ were kindly provided by Dr. D. R. Trentham (Division of Physical Biochemistry, NIMR, London). Collagenase was obtained from Worthington, thapsigargin from Alomone Laboratories (Jerusalem, Israel), 2,5-di-tert-butylhydroquinone from Aldrich-Chemie, and all other reagents from Sigma or Roche Molecular Biochemicals.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of Ca²⁺-Mg²⁺-ATPase Inhibitors on Ca²⁺ Mobilization in Single Liver Cells-- The perfusion of a single guinea pig hepatocyte with tBuBHQ (50 µM) or thapsigargin (3 µM) led to a slow rise in Ca²⁺-dependent K⁺ permeability (Fig. 1), even in the absence of external Ca²⁺. The response to the Ca²⁺-Mg²⁺-ATPase inhibitors rapidly decreased to the resting levels in the absence of external Ca²⁺ but remained at an intermediate level in the presence of external Ca²⁺ (data not shown). After the initial response to tBuBHQ or thapsigargin in the absence of external Ca²⁺, norepinephrine (5 µM) was still able to induce a large rise in Ca²⁺-dependent K⁺ permeability (Fig. 1). The signal was often larger in amplitude than that evoked by the Ca²⁺-Mg²⁺-ATPase inhibitors alone, indicating that the maximal concentrations of the inhibitors did not release all the Ca²⁺ present in the InsP₃-sensitive Ca²⁺ stores in these cells. We investigated whether the InsP₃-sensitive Ca²⁺ stores were completely depleted after the decline of the response to norepinephrine in cells incubated in the presence of the Ca²⁺-Mg²⁺-ATPase inhibitors and calcium-free solutions by photoreleasing InsP₃ from a caged precursor loaded into the cell via the patch pipette. In these conditions, the release of InsP₃ induced a large rise in Ca²⁺-dependent K⁺ permeability in 10 of 15 cells tested (Fig. 1A). Therefore, the internal Ca²⁺ stores were not yet completely depleted in two-thirds of the cells tested, even after 10 min of treatment with Ca²⁺-Mg²⁺-ATPase inhibitors in the absence of external calcium and after the decline of the noradrenaline-evoked response. However, the photolytic release of InsP₃ was sufficient to empty the intracellular Ca²⁺ stores.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Single voltage-clamped guinea pig hepatocytes loaded with 10 µM caged InsP3. Sequential applications of tBuBHQ (50 µM) and norepinephrine (5 µM) applied at the horizontal bars in an external Ca2+-free solution. The photolytic release of 1 µM InsP3 (vertical arrow) had no effect on cell A and evoked an increase in Ca2+-dependent K+ current in cell B. Holding potential was 0 mV.

Store-operated Ca²⁺ Entry-- We investigated whether store-activated calcium entry occurred in guinea pig liver cells. Incubation of the cells in the absence of external Ca²⁺ for up to 10 min did not increase Ca²⁺ entry upon the addition of Ca²⁺ to the perfusing solution (all seven cells tested) (Fig. 2A). This clearly indicates that the removal of extracellular Ca²⁺ alone does not activate Ca²⁺ entry if Ca²⁺ is added back to the perfusing solution.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Evidence of Ca2+ entry activated following the depletion of intracellular InsP3-sensitive Ca2+ stores in single guinea pig hepatocytes. A, control experiment shows the absence of activated Ca2+ entry if the cell was incubated in the absence of external Ca2+ for 6 min at 40 mV. B, after Ca2+ depletion was achieved by successive applications of 50 µM tBuBHQ and 5 µM norepinephrine in external Ca2+-free solution at 40 mV, 1.8 mM Ca2+ was added to the external medium to activate capacitative Ca2+ entry. Voltage step to +40 mV significantly decreased Ca2+ influx. C, after Ca2+ depletion was achieved by successive applications of 50 µM tBuBHQ and 5 µM norepinephrine in external Ca2+-free solution, the membrane potential was stepped to +40 mV, and 1.8 mM Ca2+ was added to the external medium. The photolytic release of 2 µM InsP3 at the vertical arrow did not activate intracellular Ca2+ release or Ca2+ entry. Voltage step to 40 mV resulted in strong activation of Ca2+ influx.

The effect on Ca²⁺ entry of store depletion was tested on guinea pig hepatocytes. To empty intracellular Ca²⁺ stores completely, cells were treated with tBuBHQ (50 µM) and norepinephrine (5 µM) as in Fig. 1. After the norepinephrine-evoked response, the agonist was washed out, and the extent Ca²⁺ store depletion was assessed by photolytic release of InsP₃. The current responses to voltage steps from 0 mV to 40 or +40 mV in the absence of external Ca²⁺ were identical (data not shown). In contrast, in the presence of external Ca²⁺, a large outward current developed (15 of 24 cells tested) if cell potential was held at 40 mV (Fig. 2B). At this potential, the Ca²⁺ driving force is increased and a higher level of Ca²⁺ entry, as shown by the large K⁺ current, was observed. Voltage step from 40 to +40 mV resulted in the biphasic behavior of the outward K⁺ current (Fig. 2B). An initial and transient increase in K⁺ current was observed due to an approximate tripling of the K⁺ driving force at +40 mV. The amplitude of the K⁺ current then decreased due to a decrease in Ca²⁺ entry at +40 mV concomitant with a large reduction in free Ca²⁺ concentration close to the membrane as Ca²⁺ was extruded from the cell by the plasma membrane Ca²⁺-Mg²⁺-ATPase. Since the current/voltage relationship of the Ca²⁺-dependent K⁺ conductance is linear (15, 16), a reduction in K⁺ current between 40 mV and +40 mV and the calculated reverse potential for Ca²⁺ in our recording conditions can only reflect a decrease in Ca²⁺ entry. This was confirmed by an experiment showing that adding 1.8 mM Ca²⁺ back to the external solution if the membrane potential was first held at +40 mV had no effect (Fig. 2C). No activation of the K⁺ current was observed after the photolytic release of 2 µM InsP₃, indicating that the stores remained empty. Voltage step to 40 mV evoked a high level of Ca²⁺ entry, since the Ca²⁺ driving force was much greater. These results show that the depletion of intracellular Ca²⁺ stores in hepatocytes is essential to trigger the opening of a Ca²⁺-permeant pathway through the plasma membrane.

Norepinephrine-evoked Ca²⁺ Influx-- The effects of Ca²⁺-mobilizing agonists on store-operated Ca²⁺ entry were investigated. Assuming that Ca²⁺ store depletion was complete after treatment with tBuBHQ (50 µM) and norepinephrine (5 µM) in cells that did not respond to photoreleased InsP₃, we compared the effects of reintroducing Ca²⁺ to the external medium in the presence or the absence of a Ca²⁺-mobilizing agonist (Fig. 3). Norepinephrine (5 µM) consistently had no effect until 1.8 mM Ca²⁺ was added back to the external medium, resulting in a large activation of K⁺ conductance, reflecting a high level of Ca²⁺ entry. The K⁺ conductance declined to initial levels if Ca²⁺ and norepinephrine were washed away. The same applications were then tested in reverse order on the same cell. The reintroduction of extracellular Ca²⁺ alone resulted in a Ca²⁺ entry that was greatly increased by the addition of 5 µM norepinephrine (Fig. 3). Similar results were obtained in 10 experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Norepinephrine potentiates Ca2+ entry in Ca2+-depleted single guinea pig hepatocytes. After depletion of the intracellular InsP3-sensitive Ca2+ stores by successive applications of 50 µM tBuBHQ and 5 µM norepinephrine in external Ca2+-free solution, a further addition of norepinephrine (5 µM) did not activate the Ca2+-dependent K+ current until 1.8 mM Ca2+ was added back to the external medium. The second part of the trace (continuous recording) shows the additional effect of norepinephrine after the reintroduction of Ca2+ to the external medium for the same cell. Holding potential was 40 mV.

InsP₃-evoked Ca²⁺ Influx-- Since norepinephrine increases Ca²⁺ entry, we tested the effects of InsP₃ on store-operated Ca²⁺ influx. Prolonged application of maximal concentrations of norepinephrine in the presence of Ca²⁺-Mg²⁺-ATPases inhibitors was not always sufficient to totally deplete Ca²⁺ from the internal stores. We therefore used externally applied monohydroxylated bile acids such as taurolithocholate sulfate (TLC-S) to deplete internal stores. These bile acids are more effective than InsP₃ for mobilizing the entire Ca²⁺ contents of the ER (25). Cells were successively treated with 10 µM norepinephrine and 200 µM TLC-S in the presence of Ca²⁺-Mg²⁺-ATPase inhibitors and in the absence of external Ca²⁺. In these conditions, the photolytic release of 2 µM InsP₃ did not evoke any rise in Ca²⁺-dependent K⁺ conductance in any of the seven cells tested (data not shown).

To test the involvement of InsP₃ in activation of the Ca²⁺ influx in Ca²⁺-depleted cells, it was necessary to release InsP₃ before the reintroduction of Ca²⁺. However, in guinea pig liver cells, InsP₃ is actively metabolized, and the response to the photolytic release of InsP₃ is short lived (10-20 s) in guinea pig liver cells in normal conditions (17). This problem was overcome by means of photolytic release of a stable analog of InsP₃, 5-thio-InsP₃, which is resistant to InsP₃ 5-phosphatase but not to InsP₃ 3-kinase (26). 5-thio-InsP₃ was photolytically released from a caged precursor introduced into the cell via the patch pipette. The intracellular Ca²⁺ stores were emptied by successive applications of norepinephrine (10 µM) and TLC-S (200 µM) in the presence of either 50 µM tBuBHQ or 3 µM thapsigargin in Ca²⁺-free solutions. In these conditions, the reintroduction of Ca²⁺ ions to the external medium induced Ca²⁺ entry (Fig. 4). Ca²⁺ ions were then washed away, but, since Ca²⁺ could have refilled the stores during this first application, TLC-S was reapplied for 1 min (not shown). Twenty µM 5-thio-InsP₃, a concentration that has been shown to evoke a high level of Ca²⁺ release in normal nondepleted guinea pig liver cells (24), was then photolytically released in the absence of external Ca²⁺. After a delay of 5 s without response, perfusion of the cell with a regular Ca²⁺-containing solution induced a much larger and faster increase in Ca²⁺-dependent K⁺ conductance, reflecting a higher level of Ca²⁺ influx (Fig. 4). All four cells tested showed an enhanced response in the presence of 5-thio-InsP₃. This provides evidence for a strong link between the activation of InsP₃ receptors and Ca²⁺ entry.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Potentiation of Ca2+ entry by a stable analog of InsP3 following depletion of intracellular InsP3-sensitive Ca2+ stores by successive applications of 3 µM thapsigargin, 10 µM norepinephrine, and 200 µM taurolithocholate sulfate in external Ca2+-free solution (not shown) to a single guinea pig hepatocyte. Superimposed traces from the same cell show successive application of 1.8 mM extracellular Ca2+ in the presence or absence 20 µM 5-thio-InsP3. The bottom trace (control) shows activation of capacitative Ca2+ entry after reintroduction of 1.8 mM Ca2+ (vertical arrow, Ca) into the external medium. Ca2+ was washed off between the two applications, and 200 µM taurolithocholate was perfused for 1 min. Since intracellular Ca2+ stores were empty, the photolytic release of 5-thio-InsP3 (vertical arrow, F) had no effect until 1.8 mM Ca2+ was added to the medium. The cell was loaded with 180 µM caged 5-thio-InsP3, and membrane potential was held at 40 mV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ store depletion is believed to be a crucial step for Ca²⁺ influx. Most of the experiments described here were performed with Ca²⁺ stores totally depleted, but in preliminary experiments, even partial Ca²⁺ depletion triggered a Ca²⁺ influx in 62% of the cells tested (n = 24). Indeed, we found that total depletion of the InsP₃-sensitive Ca²⁺ stores was essential for observation of the potentiating effect of InsP₃ on this type of Ca²⁺ entry. Since InsP₃ is unlikely to directly open a Ca²⁺ channel in the plasma membrane, our results are consistent with recent reports showing a direct coupling between InsP₃ receptors and TRP (3-5). It has been known for several years that the plasma membrane and internal Ca²⁺ stores are tightly associated (27-29). Dissociation of this tight link by the stimulation of actin polymerization seems to prevent capacitative Ca²⁺ entry in A7r5 smooth muscle cells (30).

A previous report showed that vasopressin and Ca²⁺ depletion activated two distinct pathways of Ca²⁺ entry in rat hepatocytes (10). Our results demonstrate that InsP₃ is involved in this process. In light of recent studies (3-5), we suggest that InsP₃ is required to open Ca²⁺ channels in the plasma membrane, although it does not do this by direct interaction. The reintroduction of Ca²⁺ to the external medium had an immediate effect on activation of the Ca²⁺-dependent K⁺ conductance, providing additional evidence for the existence of store-operated Ca²⁺ channels in the plasma membrane. We cannot draw firm conclusions about the nature of the channels involved in this type of Ca²⁺ entry, since reports have shown TRP to be absent in the adult liver (6). However, it is possible that this tissue may have a specific as yet uncharacterized TRP subtype.

The transient nature of the responses to 5-thio-InsP₃ may reflect its partial degradation by InsP₃ 3-kinase (26), desensitization of the InsP₃-evoked response as observed at constant InsP₃ concentration (24), rapid inactivation of I_crac (31), or the periodic store-independent inactivation of store-operated Ca²⁺ channels (32). The effect of store depletion on Ca²⁺ permeability was sustained. Therefore, the transient nature of activation by InsP₃ probably reflects either degradation or desensitization.

Direct evidence that inositol phosphates affect the activation of Ca²⁺ influx is rare. The presence of InsP₃-activated Ca²⁺ channels was first shown in the excised plasma membrane of T lymphocytes (33). We cannot exclude the involvement of InsP₄ in the responses observed, since it has been shown that 5-thio-InsP₃ may act as a substrate for InsP₃ 3-kinase (26). InsP₄ also activates Ca²⁺ channels in the plasma membrane of A431 endothelial cells (34). Although they are more active at high [Ca²⁺]_i, the activity of these InsP₄-sensitive Ca²⁺ channels at resting [Ca²⁺]_i may be sufficient to evoke the increase in Ca²⁺ influx observed in our experiments. The photolytic release of InsP₄ from caged InsP₄ should allow us to test this specific point.

	FOOTNOTES

^* This work was supported by Association pour la Recherche sur le Cancer Grant 5241 (to T. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Singer-Polignac sponsorship. Present address: INSERM U533, Hôtel-Dieu, 44000 Nantes, France.

^§ Present address: EA 2674, Université de Nice-Sophia Antipolis, Faculté des sciences, Parc Valrose, 06108 Nice, France.

^¶ To whom correspondence and reprint requests should be addressed. Tel.: 33-169156865; Fax: 33-169155893; E-mail: thierry.capiod@ibaic.u-psud.fr.

	ABBREVIATIONS

The abbreviations used are: InsP₃, inositol 1,4,5-trisphosphate; InsP₄, inositol 1,3,4,5-tetrakisphosphate; tBuBHQ, 2,5-di(tert-butyl)-1,4-benzohydroquinone; TRP, transient receptor potential; TLC-S, taurolithocholate sulfate; 5-thio-InsP₃, 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guihard, G. Articles by Capiod, T. Search for Related Content PubMed PubMed Citation Articles by Guihard, G. Articles by Capiod, T. Social Bookmarking                      What's this?